Dissociation and Culture of Adult Mouse Satellite Glial Cells

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging. Key features • Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice. • Cells are dissociated in approximately 2–3 h and cultured for approximately two weeks. • These SGC cultures allow both molecular and functional studies.

This protocol is used in: Brain, Behavior, and Immunity (2023), DOI: 10.1016/j.bbi.2023.08.005Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells.They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system.Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection.In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain.Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently.This has impeded further studies of their function and the testing of new therapeutics.This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity.We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging.

Solutions
Note: Prepare all solutions in a laminar flow cell culture hood.

Laboratory supplies
Published: Dec 20, 2023 5. Next, use scissors to decapitate the mice in a sink for the purpose of euthanasia and blood removal.This will facilitate the tissue dissection process.

B. Tissue dissection
1. Move the mouse to a designed tissue dissection area and liberally spray the back fur with 70% ethanol.2. To expose the back muscles, make a large transverse cut in the middle of the back skin using standard scissors.Then, pull the skin in opposite directions to remove all of the back skin.3. To remove the back muscles, make two long cuts close to the left and right sides of the spinal column.
Then, use forceps to pull away the muscles starting from the rostral end.4. To expose the spinal cord and DRGs, perform a laminectomy by removing the top of the vertebral canal.
Use spring scissors to cut the bones on both sides of the vertebral canal in a 45° angle to avoid damaging or losing the DRGs.Continue cutting from side to side while lifting the top of the vertebral canal, starting from the rostral end and working your way down to the caudal end of the spinal column.5. Next, use forceps to slowly remove the spinal cord in a rostral to caudal direction from the column and expose the DRGs.Discard the spinal cord.The DRGs are located in the dorso-lateral position of the vertebral canal and can be recognized by their round shape and hyaline appearance, which is different from the white color of the attached nerve fibers (Supplementary Figure 1).

C. Cell dissociation
1. Centrifuge the microtubes containing the cleaned DRGs at 200× g for 1 min and remove the HBSS dissociation solution.2. Perform the first enzymatic dissociation by adding 1.5 mL of papain solution into the microtubes.Gently flick the microtubes to resuspend the DRGs and incubate them for 20 min in a 37 °C water bath.Note: Do not resuspend with a pipette or vigorously shake to maintain the integrity of the cells.3. Centrifuge the microtubes at 200× g for 1 min and remove the papain solution.4. Wash the DRGs with 1 mL of HBSS dissociation solution, centrifuge (200× g for 1 min), and remove the solution.5. Perform the second enzymatic dissociation by adding 1.5 mL of collagenase solution into the microtubes.
Gently flick the microtubes to resuspend the DRGs and incubate them for 20 min in a 37 °C still water bath.Note: Do not resuspend with the pipette or vigorously shake to maintain the integrity of the cells.6. Centrifuge the microtubes at 200× g for 1 min and remove all the collagenase solution.
Note: From this point forward, the opening of tubes/plates that contain any tissue, cells, media, or reagents should be done in a laminar flow cell culture hood. 7. Resuspend the DRGs in 1 mL of DMEM culture medium.Centrifuge at 200× g for 1 min and carefully remove all culture solution without disturbing the tissue.8. Resuspend the DRGs in 0.5 mL of DMEM culture medium.Perform mechanical dissociation by trituration using a P1000 pipette until the solution becomes cloudy (approximately 15-20 times up and down).During trituration, the tissues should pass through the tip with a little friction initially and progress to passing easily.Be gentle and avoid introducing air bubbles at this step; this is critical.9. Add 1 mL of DMEM culture medium to each microtube.Next, transfer the contents of all microtubes into one 50 mL conical tube.Adjust the volume to approximately 5 mL with DMEM culture medium and gently mix the contents by flicking the tube.

D. Cell plating and growth
1. Transfer the resuspended cells into a 12-well microplate by pipetting 400 μL of the filtrate into each well containing sterilized cover glasses.Then, incubate the plate in a CO2 cell culture incubator (37 °C, 5% CO2).Note: During this incubation (24 h), due to the lack of coating of the cover glasses and use of DMEM lowglucose media, SGC cells will attach to the bottom of the dish while the DRG neurons and debris will remain in suspension.2. One day after plating the cells, replace the DMEM culture medium with 400 μL of fresh DMEM culture medium to remove cell debris.Always pre-warm the culture medium to 37 °C before use.3. SGCs proliferate and develop a spindle-shaped morphology (Figure 1) during the next 10-14 days.To maintain SGCs cultures, replace the DMEM culture medium every 2-3 days.

Validation of protocol
In our research article, we used this protocol to study the role of matrix metalloproteinases in SGCs and pain (Tonello et al., 2023).Here, we present additional evidence that this protocol is both robust and reproducible by assessing these cultures using molecular and functional approaches.

SGC cultures with an average cell purity of over 90%
We assessed the purity of cell culture using immunofluorescence, following the method described in our publication (Tonello et al., 2023).In brief, after 10-14 days, we replaced the DMEM culture medium with a 4% paraformaldehyde solution and incubated it for 15 min at room temperature (RT).Next, we rinsed the cultures with PBS and blocked them with 1% BSA and 0.2% Triton X-100 in PBS (BSA solution).We then incubated them overnight at 4 °C in the BSA solution with the anti-glutamine synthetase primary antibody (GS, rabbit, 1:5,000).Afterward, we incubated the cultures with the anti-rabbit secondary antibodies conjugated to Alexa Fluor 546 (1:1,000) for 1 h at RT in the BSA solution.Finally, we treated them with the universal nuclear staining DAPI in PBS for 1 min at RT.We then removed the cover glasses from the microplate and covered the side with the cultured SGCs with Prolong Gold Antifade Mountant, mounting them on glass microscope slides.Images were acquired using a Keyence BZ-X800 microscope, and cells were manually counted using NIH Image J open-source software (Schindelin et al., 2012).Cell purity was assessed as a percentage, calculated by dividing the number of GS+ cells (i.e., SGCs) by the number of DAPI+ cells (i.e., all cells in the culture) (Figure 1A).Our protocol resulted in SGC cultures with an average cell purity of 91.4% ± 1.1% (Figure 1B).Approximately 9% of the cells positive only for DAPI were considered potential contaminants.Fibroblasts, characterized as flat cells with a large nucleus, were identified as a potential major contaminant (Figure 1A).It should be noted that a reduced expression of GS has been reported in SGC culture (Belzer et al., 2010).Other markers of SGC, such as fatty acid binding protein 7 (Avraham et al., 2020), may be used instead.

Cultures show high expression of SGC transcriptional markers
We assessed the transcriptional expression levels of various cell markers using qPCR, following the method described in our publication (Tonello et al., 2023).In brief, after 10-14 days, we replaced the DMEM culture medium with TRIzol reagent to lyse SGCs and isolate the RNA.We extracted the total RNA from the TRIzol reagent using the Direct-zol RNA MiniPrep kit and then assessed the amount and quality using a UV-Vis spectrophotometer.
We then converted the RNA into cDNA using a high-capacity cDNA reverse transcription kit.Specific primers for various cell markers and Gapdh (used for normalization) were obtained from PrimerBank (Wang et al., 2012).The primer sequences can be found in Supplementary Table 1.qPCR was conducted using PowerUp SYBR Green Master Mix on a QuantStudio 3 Real-Time PCR System.Relative transcriptional expression ratios were calculated using the Pfaffl method (Pfaffl, 2001).

Cultures exhibit functional response to EDNRB agonist
We assessed the functional integrity of our SGC cultures using calcium imaging, following the method outlined in our publications (Lee et al., 2020;Ford et al., 2021).In brief, after 10-14 days, we replaced the DMEM culture medium and incubated the SGC culture for 45 min at room temperature with a solution of Fura2-AM (3 μg/mL) in DMEM containing 10% FBS and 1% P/S.We then removed the cover glasses from the cell culture microplates and transferred them to a recording chamber containing an external recording solution with the following concentrations: 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, and 10 mM HEPES.Illuminance signal was acquired using 365/385-nm switching LED controlled by the MetaFluor software (Molecular Devices) on an Olympus BX51 microscope.Previous research shows that endothelin-1 effectively increases intracellular calcium levels in SGCs by activating the endothelin receptor EDNRB (Feldman-Goriachnik and Hanani, 2017).Here, we show that IRL 1620 (10 nM), a potent and selective EDNRB agonist (Takai et al., 1992), induced an intracellular calcium increase in most, if not all, SGCs (Figure 3A and B).This experiment serves as a proof of concept for the functional integrity of our SGC cultures.These cultures may be used in future studies to evaluate and measure calcium responses to various agonists, including neurotransmitters or cytokines (Souza et al., 2013;Afroz et al., 2019).

General notes and troubleshooting
SGCs have been identified as a potential therapeutic target for the treatment of chronic pain.However, our understanding of SGC biology is limited due to the lack of techniques to study these cells in vitro.Here, we provide a step-by-step protocol for the dissociation and culture of adult mouse SGCs:

6 .
Using the stereomicroscope, grasp the dorsal root with #5 forceps, carefully pull it a few millimeters, and cut the spinal nerve (immediately distal to the ganglion) with spring scissors.Next, cut the dorsal root and place the collected DRGs in the culture dish on ice.Typically, an experienced experimenter can collect approximately 40 ganglia per mouse.7. If needed, move the culture dish under the stereomicroscope and use a pair of spring scissors and #5 forceps to remove any extra nerves, roots, and connective tissues from the DRGs.Then, transfer the cleaned DRGs equally into two microtubes.

Published: Dec 20, 2023 10 .
Filter the mixture using a 40 μm cell strainer followed by a 10 μm cell strainer.Collect the filtrate each time in a 50 mL tube.If necessary, adjust the volume of the filtrate to 5 mL with DMEM culture medium and resuspend the cells by gently flicking the tube.

Figure 3 .
Figure 3. Calcium responses in satellite glial cell (SGC) cultures.(A) Images of Fura-2 signaling in SGC cultures and (B) representative calcium imaging traces of SGCs (arrows) in response to the EDNRB agonist IRL 1620 (10 nM).Colors correspond to encircled regions of interest in A.